Method for the quantitative determination of D-3-hydroxybutyric acid and acetoacetic acid, and analytical reagent therefor

ABSTRACT

Disclosed is a method for the quantitative determination of D-3-hydroxybutyric acid and acetoacetic acid, which comprises reacting a biological sample containing D-3-hydroxybutyric acid and acetoacetic acid, with a reagent comprising: (1) a D-3-hydroxybutyrate dehydrogenase, (2) A 1  and (3) B 1 , the components (1), (2) and (3) participating in the following cycling reaction: ##STR1## thereby effecting the enzymatic cycling reaction, and measuring a change in the amount of A 2  formed or the amount of B 1  consumed. Also disclosed is an analytical reagent comprising the components (1), (2) and (3) for use in the above method. The method and the analytical reagent ensure rapidness and accuracy in the determination of D-3-hydroxybutyric acid and acetoacetic acid, even with the use of a small quantity of a biological sample, so that they are very useful in application fields, such as clinical diagnosis and food testing.

TECHNICAL FIELD

The present invention relates to a method for the quantitativedetermination of D-3-hydroxybutyric acid and acetoacetic acid utilizingan enzymatic cycling reaction. The present invention also relates to anovel analytical reagent for use in the above-mentioned quantitativedetermination.

BACKGROUND ART

In clinical examination, it is important to determine ketone bodies,such as D-3-hydroxybutyric acid and acetoacetic acid, as criteria fordetecting metabolic failure. In addition to the above-mentioned twotypes of ketone bodies, acetone can also be mentioned as a ketone body.However, acetone is volatile and unstable. In addition, theconcentration of acetone in blood is considerably low as compared to theconcentrations of D-3-hydroxybutyric acid and acetoacetic acid. Thismeans that a metabolic failure can be successfully detected even bydetermining only D-3-hydroxybutyric acid and acetoacetic acid amongketone bodies.

Examples of conventional methods for the determination of ketone bodiesinclude the diazonium method in which acetoacetic acid is reacted with adiazonium salt to thereby produce a hydrazo compound or an azo compound,and the absorbance of the produced hydrazo or azo compound is measured;the nitroprusside method in which acetoacetic acid and acetone arereacted with a nitroprusside reagent to thereby convert acetoacetic acidand acetone to respective colorimetrically-detectable forms; the gaschromatographic method in which D-3-hydroxybutyric acid and acetoaceticacid are converted to acetone and all of the ketone bodies arequantitatively determined in terms of acetone by gas chromatography; andthe enzymatic method.

Although the diazonium method exhibits high sensitivity, the methodrequires deprotenization of a biological sample before conducting thereaction of acetoacetic acid with a diazonium salt. Further, fordetermining D-3-hydroxybutyric acid by the diazonium method, thefollowing cumbersome operations are required. Acetoacetic acid in abiological sample is determined and then, D-3-hydroxybutyric acid in thesample is converted to acetoacetic acid by D-3-hydroxybutyratedehydrogenase, followed by measurement of the acetoacetic acid (which isconverted from the D-3-hydroxybutyric acid), thus quantitativelydetermining both of the D-3-hydroxybutyric acid and acetoacetic acid inthe sample. Then, the quantity of D-3-hydroxybutyric acid is obtained bysubtracting the quantity of acetoacetic acid from the above-mentionedtotal quantity of D-3-hydroxybutyric acid and acetoacetic acid. However,the conventional methods in which a ketone body is quantitativelydetermined by reacting the ketone body with a chemical reagent generallyhas low specificity and, therefore, the determination of a targetsubstance is likely to be influenced by other substances present in abiological sample. For example, in the diazonium method, thedetermination of acetoacetic acid in a biological sample is likely to beinfluenced by oxaloacetic acid (Clinica Chemica Acta, vol. 134,p.327-336, 1983). With respect to the nitroprussid method, this methodhas disadvantages in that not only cannot D-3-hydroxybutyric acid bedirectly detected as in the case of the diazonium method, but also themethod exhibits low sensitivity (detection sensitivity: 500 to 1000 μM)(Extra-edition of Japanese Journal of Clinical Medicine vol, 47, p.484,1989).

Further, the gas chromatographic method is extremely cumbersome and,therefore, the method is not suitable for use in clinical examination inwhich a lot of biological samples are to be tested.

With respect to the enzymatic method, there can be mentioned Williamsonmethod (Method of Enzymatic Analysis, Academic Press, New York,p.1836-1843, 1974) in which the enzymatic reaction ofD-3-hydroxybutyrate dehydrogenase (EC 1. 1. 1. 30) is utilized. In thismethod, when acetoacetic acid in a biological sample is to bedetermined, the reverse enzymatic reaction under the action ofD-3-hydroxybutyrate dehydrogenase is utilized in which acetoacetic acidand a reduced form of nicotinamide adenine dinucleotide (NAD) are,respectively, converted to D-3-hydroxybutyric acid and NAD. That is, adecrease in the amount of reduced NAD (which decrease corresponds to theamount of reduced NAD consumed in the above reverse reaction) whichdecrease is caused for a predetermined period of time is measured. Onthe other hand, when D-3-hydroxybutyric acid in a biological sample isto be determined, the forward enzymatic reaction under the action ofD-3-hydroxybutyrate dehydrogenase is utilized in whichD-3-hydroxybutyric acid and NAD are, respectively, converted toacetoacetic acid and reduced NAD. That is, an increase in the amount ofreduced NAD (which increase corresponds to the amount of reduced NADproduced in the above forward reaction) which increase is caused for apredetermined period of time is measured. A modification of theWilliamson method was also proposed.

As another example of the enzymatic method, there can be mentioned apaper strip testing for the determination of D-3-hydroxybutyric acid. Inthis method, the forward enzymatic reaction under the action ofD-3-hydroxybutyrate dehydrogenase is utilized in whichD-3-hydroxybutyric acid and NAD are, respectively, converted toacetoacetic acid and reduced NAD under the action of D-3-hydroxybutyratedehydrogenase. An increase in the amount of reduced NAD corresponds tothe amount of reduced NAD produced in the above forward enzymaticreaction. The amount of the D-3-hydroxybutyric acid can be determinedbased on the increase in the amount of reduced NAD. The reduced NADobtained in the above enzymatic reaction is reacted with a tetrazoliumsalt on a strip paper, and then a formazan dye produced in the amountwhich is proportional to the increase in the amount of the reduced NADis determined.

In the above-mentioned enzymatic methods, it is impossible not only todetermine both of D-3-hydroxybutyric acid and acetoacetic acid by asingle enzymatic reaction, which are clinically important ketone bodies,but also to achieve the determination with high sensitivity.

As still another example of the enzymatic method for the quantitativedetermination of acetoacetic acid, there can be mentioned a method inwhich acetoacetic acid is converted to acetyl-CoA by usingacetoacetyl-CoA synthetase (EC 6.2.1.16) and 3-ketoacyl-CoA thiolase (EC2.3.1.16) and then, the acetyl-CoA produced is used for acetylatinganiline under the action of arylamine acetylase (EC 2.3.1.5), followedby measurement of a decrease in the amount of acetylated aniline as adecrease in the absorbance at a wavelength of 405 nm (Acta Biochim.Biophys. Acad. Sci. Hung., vol. 7, p.143, 1972). However, this methodrequires complicated operations. In addition, a highly sensitive methodfor the determination of ketone bodies cannot be achieved by thismethod. Therefore, the method has not yet been widely used.

Task to be solved by the invention

As mentioned above, various types of methods for the determination ofD-3-hydroxybutyric acid or acetoacetic acid have been proposed. However,as mentioned above, in any of these methods, it is impossible todetermine ketone bodies with high sensitivity. In addition, a totalquantity of ketone bodies, which are important in clinical examination,cannot be determined by a single enzymatic reaction.

The normal concentrations of ketone bodies in serum or plasma is low.For example, in serum or plasma, the normal concentration of acetoaceticacid is as low as 41 ±1.4 (average value ±standard error) μmol/liter,the normal concentration of D-3-hydroxybutyric acid is 34 ±2.1μmol/liter, and the total normal concentration of ketone bodies is 74±2.4 μmol/liter (Extra-edition of Japanese Journal of Clinical Medicine,vol. 47, p.482, 1989). In the above situation, a highly sensitive methodfor the determination of D-3-hydroxybutyric acid ad acetoacetic acid hasbeen desired.

Means to Solve the Task

From the above context, the present inventors have made extensive andintensive studies with a view toward solving the above-mentionedproblems accompanying the conventional methods for the quantitativedetermination of a ketone body in biological samples. As a result, theyhave found that the target chemical substance, i.e., at least one ketonebody selected from the group consisting of D-3-hydroxybutyric acid andacetoacetic acid can be quantitatively determined by measuring a changein amount of A₂ or B₁, which is caused by the cycling reactionrepresented by the following formula (I): ##STR2## wherein A₁ is athio-NADP compound, a thio-NAD compound, an NADP compound or anNAD-compound; A₂ is a reduced form of A₁ ; B₁ is a reduced NADP compoundor a reduced NAD compound when A₁ is a thio-NADP compound or a thio-NADcompound, or a reduced thio-NADP compound or a reduced thio-NAD compoundwhen A₁ is an NADP compound or an NAD compound; and B₂ is an oxidizedform of B₁. The present inventors have also found that the change in theamount of coenzyme A₂ or B₁ can be easily measured because coenzymes A₂and B₁ are different in absorption maximum from each other (a reducedthio-NAD compound and a reduced thio-NADP compound exhibit an absorptionmaximum at about 400 nm, and a reduced NAD compound and a reduced NADPcompound exhibit an absorption maximum at about 340 nm). Thus, a highlysensitive method for the quantitative determination of at least oneketone body selected from the group consisting of D-3-hydroxybutyricacid and acetoacetic acid, which can be simply, efficiently carried outand is less influenced by other substances present in a sample, has beenrealized. Based upon the above findings, the present invention has beencompleted.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a graph showing the results of the rate assay ofD-3-hydroxybutyric acid at a wavelength of 400 nm conducted in Example1;

FIG. 2 is a graph showing the results of the rate assay of acetoaceticacid at a wavelength of 400 nm conducted in Example 2;

FIG. 3 is a graph showing the results of the rate assay of acetoaceticacid at a wavelength of 340 nm conducted in Example 5; and

FIG. 4 is a graph showing the results of the rate assay ofD-3-hydroxybutyric acid at a wavelength of 340 nm conducted in Example6.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided a method for thequantitative determination of at least one ketone body selected from thegroup consisting of D-3-hydroxybutyric acid and acetoacetic acid, whichcomprises:

reacting a biological sample containing at least one ketone bodyselected from the group consisting of D-3-hydroxybutyric acid andacetoacetic acid with a reagent comprising:

(1) a D-3-hydroxybutyrate dehydrogenase which utilizes the followingcoenzymes (i) and (ii):

(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and

(ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound),

and catalyzes the reversible reaction producing acetoacetic acid fromD-3-hydroxybutyric acid as a substrate;

(2) A₁ (defined hereinbelow); and

(3) B₁ (defined hereinbelow):

the components (1), (2) and (3) participating in the following cyclingreaction: ##STR3## wherein A₁ is a thio-NADP compound, a thio-NADcompound, an NADP compound or an NAD compound; A₂ is a reduced form ofA₁ ; B₁ is a reduced NADP compound or a reduced NAD compound when A₁ isa thio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; and B₂ is an oxidized form of B₁, thereby effecting thecycling reaction; and

measuring a change in the amount of A₂ or B₁.

In another aspect of the present invention, there is provided a methodfor the quantitative determination of at least one ketone body selectedfrom the group consisting of D-3-hydroxybutyric acid and acetoaceticacid, which comprises:

reacting a biological sample containing at least one ketone bodyselected from the group consisting of D-3-hydroxybutyric acid andacetoacetic acid with a reagent comprising:

(1) a D-3-hydroxybutyrate dehydrogenase as a first dehydrogenase whichutilizes the following coenzymes (i) and (ii):

(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and

(ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound),

and catalyzes the reversible reaction producing acetoacetic acid fromD-3-hydroxybutyric acid as a substrate;

(2) A₁ (defined hereinbelow);

(3) at least one coenzyme selected from, B₁ (defined hereinbelow) and B₂(defined hereinbelow); and

(4) a second dehydrogenase which does not react with D-3-hydroxybutyricacid but acts to promote the reaction for converting B₂ to B₁ in thefollowing cycling reaction, in combination with a substrate for thesecond dehydrogenase:

the components (1), (2), (3) and (4) participating in the followingcycling reaction: ##STR4## wherein A₁ is a thio-NADP compound, athio-NAD compound, an NADP compound or an NAD compound; A₂ is a reducedform of A₁ ; B₁ is a reduced NADP compound or a reduced NAD compoundwhen A₁ is a thio-NADP compound or a thio-NAD compound, or a reducedthio-NADP compound or a reduced thio-NAD compound when A₁ is an NADPcompound or an NAD compound; B₂ is an oxidized form of B₁ ; and B₂ →B₁represents an enzymatic reaction producing B₁ from coenzyme B₂ under theaction of the second dehydrogenase, thereby effecting the cyclingreaction; and

measuring a change in the amount of A₂.

In a further aspect of the present invention, there is provided a methodfor the quantitative determination of at least one ketone body selectedfrom the group consisting of D-3-hydroxybutyric acid and acetoaceticacid, which comprises:

reacting a biological sample containing at least one ketone bodyselected from the group consisting of D-3-hydroxybutyric acid andacetoacetic acid with a reagent comprising:

(1) a D-3-hydroxybutyrate dehydrogenase as a first dehydrogenase whichutilizes the following coenzymes (i) and (ii):

(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and

(ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound),

and catalyzes the reversible reaction producing acetoacetic acid fromD-3-hydroxybutyric acid as a substrate;

(2) at least one coenzyme selected from A₁ (defined hereinbelow) and A₂(defined hereinbelow); and

(3) B₁ (defined hereinbelow);

(5) a third dehydrogenase which does not react with D-3-hydroxybutyricacid but acts to promote the reaction for converting A₂ to A₁ in thefollowing cycling reaction, in combination with a substrate for thethird dehydrogenase:

the components (1), (2), (3) and (5) participating in the followingcycling reaction: ##STR5## wherein A₁ is a thio-NADP compound, athio-NAD compound, an NADP compound or an NAD compound; A₂ is a reducedform of A₁ ; B₁ is a reduced NADP compound or a reduced NAD compoundwhen A₁ is a thio-NADP compound or a thio-NAD compound, or a reducedthio-NADP compound or a reduced thio-NAD compound when A₁ is an NADPcompound or an NAD compound; B₂ is an oxidized form of B₁ ; and A₂ →A₁represents an enzymatic reaction producing A₁ from coenzyme A₂ under theaction of the third dehydrogenase, thereby effecting the cyclingreaction; and

measuring a change in the amount of B₁.

In still a further aspect of the present invention, there is provided ananalytical reagent for use in the quantitative determination of at leastone ketone body selected from the group consisting of D-3-hydroxybutyricacid and acetoacetic acid, which comprises:

(1) a D-3-hydroxybutyrate dehydrogenase which utilizes the followingcoenzymes (i) and (ii):

(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and

(ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound),

and catalyzes the reversible reaction producing acetoacetic acid fromD-3-hydroxybutyric acid as a substrate;

(2) A₁ (defined hereinbelow); and

(3) B₁ (defined hereinbelow),

wherein A₁ is a thio-NADP compound, a thio-NAD compound, an NADPcompound or an NAD compound; B₁ is a reduced NADP compound or a reducedNAD compound when A₁ is a thio-NADP compound or a thio-NAD compound, ora reduced thio-NADP compound or a reduced thio-NAD compound when A₁ isan NADP compound or an NAD compound.

In still a further aspect of the present invention, there is provided ananalytical reagent for use in the quantitative determination of at leastone ketone body selected from the group consisting of D-3-hydroxybutyricacid and acetoacetic acid, which comprises:

(1) a D-3-hydroxybutyrate dehydrogenase as a first dehydrogenase whichutilizes the following coenzymes (i) and (ii):

(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and

(ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound),

and catalyzes the reversible reaction producing acetoacetic acid fromD-3-hydroxybutyric acid as a substrate;

(2) A₁ (defined hereinbelow);

(3) at least one coenzyme selected from B₁ (defined hereinbelow) and B₂(defined hereinbelow); and

(4) a second dehydrogenase which does not react with D-3-hydroxybutyricacid but acts to promote the reaction for converting B₂ to B₁, incombination with a substrate for the second dehydrogenase,

wherein A₁ is a thio-NADP compound, a thio-NAD compound, an NADPcompound or an NAD compound; B₁ is a reduced NADP compound or a reducedNAD compound when A₁ is a thio-NADP compound or a thio-NAD compound, ora reduced thio-NADP compound or a reduced thio-NAD compound when A₁ isan NADP compound or an NAD compound; B₂ is an oxidized form of B₁.

In still a further aspect of the present invention, there is provided ananalytical reagent for use in the quantitative determination of at leastone ketone body selected from the group consisting of D-3-hydroxybutyricacid and acetoacetic acid, which comprises:

(1) a D-3-hydroxybutyrate dehydrogenase as a first dehydrogenase whichutilizes the following coenzymes (i) and (ii):

(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and

(ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound),

and catalyzes the reversible reaction producing acetoacetic acid fromD-3-hydroxybutyric acid as a substrate;

(2) at least one coenzyme selected from A₁ (defined hereinbelow) and A₂(defined hereinbelow); and

(3) B₁ (defined hereinbelow);

(5) a third dehydrogenase which does not react with D-3-hydroxybutyricacid but acts to promote the reaction for converting A₂ to A₁, incombination with a substrate for the third dehydrogenase,

wherein A₁ is a thio-NADP compound, a thio-NAD compound, an NADPcompound or an NAD compound; B₁ is a reduced NADP compound or a reducedNAD compound when A₁ is a thio-NADP compound or a thio-NAD compound, ora reduced thio-NADP compound or a reduced thio-NAD compound when A₁ isan NADP compound or an NAD compound; A₂ is a reduced form of A₁.

The D-3-hydroxybutyrate dehydrogenase to be used in the method of thepresent invention is defined as a dehydrogenase which utilizes (i) afirst coenzyme selected from the group consisting of a thio-NADPcompound and a thio-NAD compound, and (ii) a second coenzyme selectedfrom an NADP compound and an NAD compound, and catalyzes the reversiblereaction producing acetoacetic acid from D-3-hydroxybutyric acid as asubstrate. A typical example of he above-mentioned reversible reactionis represented by the following formula:

    D-3-hydroxybutyric acid +NAD(P).sup.+ ⃡acetoacetic acid +NAD(P)H +H.sup.+.

Specific examples of such D-3-hydroxybutyrate dehydrogenases as definedabove include: those derived from Pseudomonas sp., (manufactured byToyobo Co., Ltd., Japan), Rhodopseudomonas spheroides, Rhodospirillumrubrum, Pseudomonas lemoignei and animal tissues, e.g., mitochondriaderived from rat liver (see, Enzyme catalogue, No. 350.HBD, Toyobo Co.,Ltd., Japan; Enzyme Handbook, p.11-12, Asakura Shoten, Japan, 1982;Biochem. J., vol. 102, p.423-431, 1967; J. Biol. Chem., vol. 237,p.603-607, 1962; and Methods in Enzymol., vol. 14, p.227-231, Japan,1969). Among these, a D-3-hydroxybutyrate dehydrogenase derived fromPseudomonas sp. which can be used in combination with either a (thio)NADcompound or a (thio)NADP compound as a coenzyme, is especiallypreferable. With respect to the D-3-hydroxybutyrate dehydrogenasederived from Pseudomonas sp., when the activity exerted with use of NADis taken as 100%, the relative activity exerted with use of NADP is4.74% (Enzyme Catalogue, No. 350.HBD, Toyobo Co., Ltd., Japan) and therelative activity exerted with use of thio-NAD is about 10%; and whenthe activity exerted with use of NADP is taken as 100%, the relativeactivity exerted with use of thio-NADP is about 10%. With respect to theD-3-hydroxybutyrate dehydrogenases of other origins, many of theseenzymes are used in combination with an NAD compound and a thio-NADcompound. Such dehydrogenases also can be used in appropriate systems.

In the present invention, coenzymes A₁ and B₂ are appropriately selectedfrom the group consisting of a thio-NADP compound, a thio-NAD compound,an NADP compound and an NAD compound in accordance with the designedreaction scheme. Examples of thio-NADP compounds and thio-NAD compoundsinclude thionicotinamide adenine dinucleotide phosphate (thio-NADP) andthionicotinamide hypoxanthine dinucleotide phosphate; andthionicotinamide adenine dinucleotide (thio-NAD) and thionicotinamidehypoxanthine dinucleotide. Examples of NADP compounds and NAD compoundsinclude nicotinamide adenine dinucleotide phosphate (NADP),acetylpyridine adenine dinucleotide phosphate (acetyl NADP),acetylpyridine hypoxanthine dinucleotide phosphate, nicotinamidehypoxanthine dinucleotide phosphate (deamino NADP); and nicotinamideadenine dinucleotide (NAD), acetylpyridine adenine dinucleotide (acetylNAD), acetylpyridine hypoxanthine dinucleotide and nicotinamidehypoxanthine dinucleotide (deamino NAD). Hereinafter, reduced types ofthese coenzymes are referred to, for example, as a thio-NADPH compound,a thio-NADH compound, an NADPH compound and an NADH compound.

In the present invention, for example, when A₁ is a thio-NAD(P)compound, B₁ is an NAD(P)H compound, and when A₁ is an NAD(P) compound,B₁ is a thio-NAD(P)H compound.

When the D-3-hydroxybutyrate dehydrogenase to be used is capable ofreacting only with NAD type coenzymes, i.e., a thio-NAD compound and anNAD compound, appropriate NAD type coenzymes are selected from theabove-mentioned thio-NAD compounds and NAD compounds. When theD-3-hydroxybutyrate dehydrogenase to be used is capable of reacting onlywith NADP type coenzymes, i.e., a thio-NADP compound and an NADPcompound, appropriate NADP coenzymes are selected from theabove-mentioned thio-NADP compounds and NADP compounds. When theD-3-hydroxybutyrate dehydrogenase to be used is capable of reacting witheither NAD type coenzymes or NADP type coenzymes, appropriate NAD typeor NADP type coenzymes are selected from the above-mentioned thio-NADcompounds, thio-NADP compounds, NAD compounds and NADP compounds.

In the method of the present invention, component (2) (coenzyme A₁) andcomponent (3) (coenzyme B₁) are used in excess amounts relative not onlyto the total amount of D-3-hydroxybutyric acid and acetoacetic acid, butalso the respective Km (Michaelis constant) values of component (1)(D-3-hydroxybutyrate dehydrogenase) for components (2) and (3). It isespecially preferred to use each of components (2) and (3) in a molaramount which is 20 to 10,000 times the total mole of D-3-hy droxybutyricacid and acetoacetic acid.

As mentioned above, in another aspect of the present invention, there isprovided an analytical reagent for use in the above-mentionedquantitative determination. In the analytical reagent of the presentinvention, the concentration of each of component (2) (A₁) and component(3) (B₁) is 0.02 to 100 mM, preferably 0.05 to 20 mM, and theconcentration of component (1) (D-3-hydroxybutyrate dehydrogenase) is 5to 1,000 U/ml, preferably 20 to 400 U/ml. However, an appropriateconcentration of each of components (1), (2) and (3) in the analyticalreagent is varied depending on the type of a biological sample to betested and, if desired, these components can be used in larger amounts.

In the present invention, when B₂ functions also as a coenzyme for acertain dehydrogenase other than the D-3-hydroxybutyrate dehydrogenaseof the cycling reaction (I), which certain dehydrogenase does not reactwith D-3-hydroxybutyric acid but acts to promote the reaction forconverting B₂ to B₁ in cooperation with a substrate for the certaindehydrogenase, such a certain dehydrogenase (which is hereinafterfrequently referred to as "second dehydrogenase") can also beadditionally incorporated together with a substrate therefor (seconddehydrogenase is referred to also as "component (4)") into the reagentfor effecting the following cycling reaction (II): ##STR6## wherein A₁is a thio-NADP compound, a thio-NAD compound, an NADP compound or an NADcompound; A₂ is a reduced form of A₁ ; B₁ is a reduced NADP compound ora reduced NAD compound when A₁ is a thio-NADP compound or a thio-NADcompound, or a reduced thio-NADP compound or a reduced thio-NAD compoundwhen A₁ is an NADP compound or an NAD compound; or an NAD compound; andB₂ is an oxidized form of B₁.

In the above reaction system, as the second dehydrogenase, adehydrogenase which does substantially not react with A₁ but acts topromote the reaction for converting B₂ to B₁, is chosen. Alternatively,by choosing reaction conditions under which the second dehydrogenasedoes not react with A₁, the objective can be attained. For example,there can be chosen appropriate A₁ -B₂ amount relationship conditionsunder which the second dehydrogenase does substantially not react withA₁. The quantitative determination of the target chemical substanceparticipating in reaction (II), can be achieved by measuring a change inthe amount of A₂ [which is caused for a predetermined period of timeduring reaction (II)].

The second dehydrogenase can be advantageously used in order toregenerate B₁, so that the amount of B₁ to be used in the analyticalreaction can be reduced. This is particularly useful when B₁ is anexpensive compound. It is also possible to use B₂ alone or a mixture ofB₁ and B₂ at the initiation of the reaction. For conducting reaction(II), an amount of at least one coenzyme selected from B₁ and B₂ ispreferably not larger than 1/10 mole per mole of A₁, although the amountis not particularly limited.

In practicing the above method for quantitative determination of thepresent invention using a second dehydrogenase as component (4), A₁ isused in a concentration of 0.02 to 100 mM, preferably 0.05 to 20 mM. B₂and/or B₁ is used in a concentration of 0.05 to 5,000 μM, preferably 5to 500 μM. A D-3-hydroxybutyrate dehydrogenase as the firstdehydrogenase is used in a concentration of 5 to 1000 U/ml, preferably20 to 400 U/ml. The concentration of the second dehydrogenase (U/ml) canbe 20 times or more the Km value (unit: mM) thereof for B₂, e.g., 1 to100 U/ml. The substrate for the second dehydrogenase can be used in astoichiometrically excess amount, for example, 0.05 to 20 mM. Theamounts of the components of the reagent for the cycling reaction can bevaried depending on the type of a biological sample to be tested. Theamount exceeding the above can also be employed.

As examples of combinations of second dehydrogenases and substratestherefor, the following combinations can be mentioned. When B₂ is an NADcompound or a thio-NAD compound, there can be mentioned combinations of:alcohol dehydrogenase (EC 1.1.1.1) and ethanol; glycerol dehydrogenase(EC 1.1.1.6) derived from E. coli and glycerol; glycerol-3-phosphatedehydrogenase (EC 1.1.1.8) derived from rabbit muscle andL-glycerol-3-phosphate; malate dehydrogenase (EC 1.1.1.37) derived frompig or bovine heart and L-malic acid; and glyceraldehyde phosphatedehydrogenase (EC 1.1.1.12) derived from rabbit muscle, liver, yeast orE. coli, D-glyceraldehyde phosphate and phosphoric acid. When B₂ is anNADP compound or a thio-NADP compound, there can be mentionedcombinations of: glucose-6-phosphate dehydrogenase (EC 1.1.1.49) derivedfrom yeast and D-glucose-6-phosphate; isocitrate dehydrogenase (EC1.1.1.42) derived from yeast or pig heart and isocitric acid; glyoxylatedehydrogenase (EC 1.2.1.17) derived from Pseudomonas oxalaticus, CoA andglyoxylic acid; phosphogluconate dehydrogenase (EC 1.1.1.44) derivedfrom rat liver, beer yeast or E. coli and 6-phospho-D-gluconic acid;glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13) derived from plantchlorophyll, D-glyceraldehyde-3-phosphate and phosphoric acid; andbenzaldehyde dehydrogenase (EC 1.2.1.7) derived from Pseudomonasfluorescens and benzaldehyde.

Furthermore, in the present invention, when A₂ functions also as acoenzyme for a certain dehydrogenase other than the D-3-hydroxybutyratedehydrogenase of the reaction (I) and the second dehydrogenase of thereaction (II), which certain dehydrogenase does not react withD-3-hydroxybutyric acid but acts to promote the reaction for convertingA₂ to A₁ in cooperation with a substrate for the certain dehydrogenase,such a certain dehydrogenase (which is hereinafter frequently referredto as "third dehydrogenase") can also be additionally incorporatedtogether with a substrate therefor (third dehydrogenase is referred toas "component (5)") into the reagent for effecting the following cyclingreaction (III): ##STR7## wherein A₁ is a thio-NADP compound, a thio-NADcompound, an NADP compound or an NAD compound; A₂ is a reduced form ofA₁ ; B₁ is a reduced NADP compound or a reduced NAD compound when A₁ isa thio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; and B₂ is an oxidized form of B₁.

In the above reaction system, as the third dehydrogenase, adehydrogenase which does substantially not react with B₁ but acts topromote the reaction for converting A₂ to A₁, is chosen. Alternatively,by choosing reaction conditions under which the third dehydrogenase doesnot react with B₁, the objective can be attained. For example, there canbe chosen appropriate B₁ -A₂ amount relationship conditions under whichthe third dehydrogenase does substantially not react with B₁. Thequantitative determination of the target chemical substanceparticipating in reaction (III), can be achieved by measuring a changein the amount of B₁ [which is caused for a predetermined period of timeduring reaction (III)].

The third dehydrogenase can be advantageously used in order toregenerate A₁, so that the amount of A₁ to be used in the analyticalreaction can be reduced. This is particularly useful when A₁ is anexpensive compound. It is also possible to use A₂ alone or a mixture ofA₁ and A₂, at the initiation of the reaction. For conducting reaction(III), an amount of at least one coenzyme selected from A₁ and A₂ ispreferably not larger than 1/10 mole per mole of B₁, although the amountis not particularly limited.

In practicing the above method for quantitative determination of thepresent invention using a third dehydrogenase as component (5), B₁ isused in a concentration of 0.02 to 100 mM, preferably 0.05 to 20 mM. A₂and/or A₁ is used in a concentration of 0.05 to 5,000 μM, preferably 5to 500 μM. D-3-hydroxybutyrate dehydrogenase as the first dehydrogenaseis used in a concentration of 5 to 1000 U/ml, preferably 20 to 400 U/ml.The concentration of the third dehydrogenase (U/ml) can be 20 times ormore the Km value (unit: mM) thereof for A₂, e.g., 1 to 100 U/ml. Thesubstrate for the third dehydrogenase can be used in astoichiometrically excess amount, for example, 0.05 to 20 mM. Theamounts of the components of the reagent for the cycling reaction can bevaried depending on the type of a biological sample to be tested. Theamounts exceeding the above can also be employed.

As Examples of combinations of third dehydrogenases and substratestherefor, the following combinations can be mentioned. When A₁ is an NADcompound or a thio-NAD compound, there can be mentioned combination of:alcohol dehydrogenase (EC 1.1.1.1) and acetaldehyde; glyceroldehydrogenase (EC 1.1.1.6) derived from E. coli and dihydroxyacetone;glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) derived from rabbitmuscle and dihydroxyacetone phosphate; malate dehydrogenase (EC1.1.1.37) derived from pig or bovine heart and oxaloacetic acid; andglyceraldehyde phosphate dehydrogenase (EC 1.1.1.12) derived from rabbitmuscle, liver, yeast or E. coli and 1,3-diphospho-D-glycerate. When A₁is an NADP compound or a thio-NADP compound, there can be mentionedcombinations of: glucose-6-phosphate dehydrogenase (EC 1.1.1.49) derivedfrom yeast and D-glucono-S-lactone-6-phosphate; and glyceraldehydephosphate dehydrogenase (EC 1.2.1.13) derived from plant chlorophyll and1,3-diphospho-D-glyceric acid.

In the method of the present invention, types of coenzymes A₁ and B₁ canbe appropriately selected, taking into consideration the relativeactivities and the like of the coenzymes, and pH conditions for theforward and reverse reactions can also be appropriately selected so thatthe enzymatic cycling reaction proceeds efficiently.

In practicing the method for the quantitative determination ofD-3-hydroxybutyric acid and acetoacetic acid in a biological sample byusing the analytical reagent of the present invention, 0.001 to 1 ml ofthe biological sample can be added to an aqueous composition containingthe above-defined components (1) to (3), (1) to (4), or (1) to (3) and(5), and the resultant solution is reacted at about 37° C. Then, achange in the absorbance at a wavelength specific for coenzyme A₂ or B₁is measured, which is observed with respect to the reaction mixture asbetween predetermined two time points during the reaction (e.g., between3 and 4 minutes after the start of the reaction, or between 3 and 8minutes after the start of the reaction). The period between such twotime points during the reaction can be varied in the range from severalminutes to several tens of minutes, depending on the type of abiological sample and the type of a target chemical substance containedtherein. For example, when A₂ is a thio-NADH compound and B₁ is an NADHcompound, either the produced A₂ is determined in terms of the change inabsorbance at 400 nm, or the consumed B₁ is determined in terms of thechange in absorbance at 340 nm. The thus obtained change in absorbancereflecting the amount of the target chemical substance is applied to acalibration curve which has been prepared with respect to a standardsample containing D-3-hydroxybutyric acid or acetoacetic acid, tothereby determine the amount of the target chemical substance. By themethod of the present invention, it becomes possible to realize areal-time quantitative determination of D-3-hydroxybutyric acid andacetoacetic acid in a biological sample.

Further, according to the method of the present invention, when abiological sample to be subjected to quantitative determination is asample which usually contains D-3-hydroxybutyric acid and acetoaceticacid, for example, serum or plasma, the total amount ofD-3-hydroxybutyric acid and acetoacetic acid, i.e., the total amount ofketone bodies, can be efficiently obtained with a single operation. Anymethod achieving such excellent performances has never been known in theart. Such excellent performances have for the first time been realizedby the method of the present invention. In the method of the presentinvention, both of D-3-hydroxybutyric acid and acetoacetic acid areskillfully introduced directly to an enzymatic cycling reaction system,based on a knowledge that both of D-3-hydroxybutyric acid andacetoacetic acid are substrates for a D-3-hydroxybutyrate dehydrogenase.

Furthermore, according to the present invention, either one ofD-3-hydroxybutyric acid and acetoacetic acid can be separatelydetermined by pretreating a biological sample containing both of theabove-mentioned ketone bodies with an enzyme which is capable ofreacting only with one of the above-mentioned two types of ketone bodiesand subsequently introducing the pretreated sample to the specificenzymatic cycling reaction of the method of the present invention. Forexample, when determination of only D-3-hydroxybutyric acid amongD-3-hydroxybutyric acid and acetoacetic acid contained in a biologicalsample (e.g., serum and plasma) is intended, the sample is pretreatedwith an acetoacetate decarboxylase (EC 4.1.1.4) to convert theacetoacetic acid to acetone and carbon dioxide, thus eliminating theacetoacetic acid. Then, the D-3-hydroxybutyric acid in the biologicalsample is quantitatively determined according to the method of thepresent invention utilizing the enzymatic cycling reaction catalyzed bya D-3-hydroxybutyrate dehydrogenase. Thereafter, the acetoacetic acidcan be quantitatively determined by subtracting the amount of theD-3-hydroxybutyric acid from the previously measured total amount of theketone bodies.

Furthermore, it should be noted that the method of the present inventionis so designed that the target chemical substance (i.e., at least oneketone body selected from the group consisting of D-3-hydroxybutyricacid and acetoacetic acid) itself participates in the enzymatic cyclingreaction. Therefore, the determination of the target chemical substanceby the method of the present invention is less influenced by othersubstances present in the sample, so that the desired determination canbe easily, simply done by rate assay without requiring any blank assayof the sample.

With respect to the determination of A₂ or B₁, other known methods canbe used instead of the measurement of absorbances described above.

Best Mode for Carrying Out the Invention

Hereinbelow, the present invention will be illustrated with reference tothe following Examples, which however should not be construed aslimiting the scope of the present invention.

EXAMPLE

    ______________________________________                                        Reagent                                                                       ______________________________________                                        100  mM      Tris-HCl buffer (pH 8.5)                                         0.1  mM      reduced NAD (manufactured by Oriental Yeast                                   Co., Ltd., Japan)                                                4    mM      thio-NAD (manufactured by Sigma Co., Ltd.,                                    U.S.A.)                                                          45   U/ml    D-3-hydroxybutyrate dehydrogenase                                             (derived from Pseudomonas sp.                                                 and manufactured by Toyobo Co., Ltd., Japan)                     ______________________________________                                    

Procedure

1 ml of the above reagent was placed in each of six cuvettes and heatedto 37° C. To the respective cuvettes were individually added 20 μl eachof six types of aqueous D-3-hydroxybutyric acid solutions respectivelyhaving concentrations of 0, 10, 20, 30, 40 and 50 μM. The resultantmixtures in the respective cuvettes were individually allowed to reactat 37° C. With respect to each of respective samples taken from thereaction mixtures in the six cuvettes, absorbances at 400 nm weremeasured 2 minutes and 5 minutes after the start of the reaction, and achange in absorbance as between the above two time points was calculatedwith respect to each of the samples. Results are shown in FIG. 1, whichdemonstrates the presence of good linearity in the relationship betweenthe change (difference) in absorbance (with respect to the reactionmixture, as between 2 and 5 minutes after the start of the reaction) andthe D-3-hydroxybutyric acid concentration.

EXAMPLE 2

Substantially the same procedure was repeated as in Example 1 exceptthat aqueous acetoacetic acid solutions were used in place of theaqueous D-3-hydroxybutyric acid solutions. Results are shown in FIG. 2,which demonstrates the presence of good linearity in the relationshipbetween the change (difference) in absorbance (with respect to thereaction mixture, as between 2 and 5 minutes after the start of thereaction) and the acetoacetic acid concentration.

EXAMPLE

    ______________________________________                                        Reagent                                                                       ______________________________________                                        100  mM      Tris-HCl buffer (pH 8.5)                                         0.1  mM      reduced NAD (manufactured by Oriental Yeast                                   Co., Ltd., Japan)                                                5    mM      thio-NAD (manufactured by Sigma Co., Ltd.,                                    U.S.A.)                                                          60   U/ml    D-3-hydroxybutyrate dehydrogenase                                             (derived from Pseudomonas sp.                                                 and manufactured by Toyobo Co., Ltd., Japan)                     0.2%     Triton X-100 (manufactured by Sigma Co.,                                      Ltd., U.S.A.)                                                        ______________________________________                                    

Procedure

1 ml of the above reagent was placed in each of four cuvettes and heatedto 37° C. To the respective cuvettes were individually added 20 μl eachof four different types of sera derived from healthy human beings. Theresultant mixtures in the respective cuvettes were individually allowedto react at 37° C. With respect to each of respective samples taken fromthe reaction mixtures in the four cuvettes, absorbances at 400 nm weremeasured 2 minutes and 5 minutes after the start of the reaction, and achange in absorbance as between the above two time points was calculatedwith respect to each of the samples.

On the other hand, substantially the same procedure as mentioned abovewas repeated except that an aqueous solution having a D-3-hydroxybutyricacid concentration of 50 μM as a standard sample and distilled waterwere individually used instead of the serum samples. Absorbance valuesobtained 2 minutes and 5 minutes after the start of the reaction withrespect to the distilled water were taken as respective reagent blanksat the above two time points, and were subtracted from the absorbancevalues obtained with respect to the serum samples as well as thestandard sample. Using the absorbance data of the standard sample andthe distilled water, a calibration curve was obtained.

The change in absorbance as between the above two time points, which wasobtained with respect to each of the serum samples, was applied to theabove-mentioned calibration curve, to thereby determine the totalconcentration of the ketone bodies (D-3-hydroxybutyric acid andacetoacetic acid). Results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Total concentration of the ketone bodies (D-                                  3-hydroxybutyric acid + acetoacetic acid) (μM)                             ______________________________________                                        Serum 1                                                                              78.3                                                                   Serum 2                                                                              48.7                                                                   Serum 3                                                                              53.4                                                                   Serum 4                                                                              62.9                                                                   ______________________________________                                    

EXAMPLE

    ______________________________________                                        Reagent (I)                                                                   ______________________________________                                        10   mM      phosphate buffer (pH 6.0)                                        0.2%     Triton X-100 (manufactured by Sigma Co., Ltd.,                                U.S.A.)                                                              10   U/ml    acetoacetate decarboxylase (derived from                                      Bacillus polymyxa and manufactured by                                         Wako Pure Chemical Industries, Ltd., Japan)                      ______________________________________                                        Reagent (II)                                                                  ______________________________________                                        200  mM      Tris-HCl buffer (pH 9.0)                                         0.2  mM      reduced NAD (manufactured by Oriental Yeast                                   Co., Ltd., Japan)                                                10   mM      thio-NAD (manufactured by Sigma Co., Ltd.,                                    U.S.A.)                                                          120  U/ml    D-3-hydroxybutyrate dehydrogenase                                             (derived from Pseudomonas sp.                                                 and manufactured by Toyobo Co., Ltd., Japan)                     ______________________________________                                    

Procedure

0.45 ml of the above reagent (I) was placed in each of four cuvettes andheated to 37° C. To the respective cuvettes were individually added 20μl each of four different types of sera which were the same as thoseused in Example 3. The resultant mixtures in the respective cuvetteswere individually allowed to react at 37° C for 5 minutes, therebyeliminating acetoacetic acid originally present in the samples. 0.05 mlof 0.2N hydrochloric acid was added to each of the resultant mixture tothereby deactivate the acetoacetate decarboxylase. Then, to therespective cuvettes were individually added 0.5 ml of the above reagent(II). The resultant mixtures in the respective cuvettes wereindividually allowed to react at 37° C. With respect to each ofrespective samples taken from the mixtures in the four cuvettes,absorbances at 400 nm were measured 2 minutes and 5 minutes after thestart of the reaction, and a change in absorbance as between the abovetwo time points was calculated with respect to each of the samples.

On the other hand, substantially the same procedure as mentioned abovewas repeated except that an aqueous solution having a D-3-hydroxybutyricacid concentration of 50 μM as a standard sample and distilled waterwere individually used instead of serum samples. Absorbance valuesobtained 2 minutes and 5 minutes after the start of the reaction withrespect to the distilled water were taken as respective reagent blanksat the above two time points, and were subtracted from the absorbancevalues obtained with respect to the serum samples as well as thestandard sample. Using the absorbance data of the standard sample andthe distilled water, a calibration curve was obtained. The change inabsorbance as between 2 minutes and 5 minutes after the start of thereaction, which was obtained with respect to each of the serum samples,was applied to the above-mentioned calibration curve to therebydetermine the concentration of D-3-hydroxybutyric acid. Further, withrespect to each of the samples, the value of the above-obtainedconcentration of D-3-hydroxybutyric acid in the sample was subtractedfrom the total concentration of ketone bodies obtained in Example 3above, thereby obtaining the concentration of acetoacetic acid in thesample. Results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        D-3-hydroxy-    Acetoacetic                                                                             Total concentration                                 butyric acid    acid      of ketone bodies                                    (μM)         (μM)   (μM)                                             ______________________________________                                        Serum 1                                                                              37.4         40.9      78.3                                            Serum 2                                                                              22.5         26.2      48.7                                            Serum 3                                                                              26.1         27.3      53.4                                            Serum 4                                                                              28.0         34.9      62.9                                            ______________________________________                                    

EXAMPLE

    ______________________________________                                        Reagent                                                                       ______________________________________                                        40   mM      Na.sub.2 CO.sub.3 --NaHCO.sub.3 buffer (pH 10.0)                 20   mM      NADP (manufactured by Oriental Yeast Co.,                                     Ltd., Japan)                                                     50   mM      thio-NAD (manufactured by Sigma Co., Ltd.,                                    U.S.A.)                                                          0.4  M       ethanol                                                          30   U/ml    alcohol dehydrogenase (manufactured by                                        Oriental Yeast Co., Ltd., Japan)                                 350  U/ml    D-3-hydroxybutyrate dehydrogenase                                             (derived from Pseudomonas sp.                                                 and manufactured by Toyobo Co., Ltd., Japan)                     ______________________________________                                    

Procedure

1 ml of the above reagent was placed in each of six cuvettes. To therespective cuvettes were individually added 50 μl each of six types ofaqueous acetoacetic acid solutions respectively having concentrations of0, 20, 40, 60, 80 and 100 μM. The resultant mixtures in the respectivecuvettes were individually allowed to react at 37° C. With respect toeach of respective samples taken from the reaction mixtures in the sixcuvettes, absorbances at 340 nm were measured 3 minutes and 8 minutesafter the start of the reaction. Absorbance values obtained 3 minutesand 8 minutes after the start of the reaction with respect to the samplefrom the cuvette containing the 0 μM concentration solution were takenas reagent blanks, and subtracted from the respective absorbance valuesobtained with respect to the samples from the remaining five cuvettes,which had different acetoacetic acid concentrations of from 20 to 100 μMas mentioned above. Using the absorption data of the samples from theremaining five cuvettes and the reagent blanks, a change in absorbanceas between the above two time points was calculated with respect to eachof the samples. Results are shown in FIG. 3, which demonstrates thepresence of good linearity in the relationship between the change(difference) in absorbance (with respect to the reaction mixture, asbetween 3 and 8 minutes after the start of the reaction) and theacetoacetic acid concentration.

EXAMPLE

    ______________________________________                                        Reagent                                                                       ______________________________________                                        50   mM      Tris-HCl buffer (pH 8.0)                                         0.25 mM      reduced NAD (manufactured by Oriental Yeast                                   Co., Ltd., Japan)                                                50   mM      thio-NAD (manufactured by Sigma Co., Ltd.,                                    U.S.A.)                                                          5    mM      dihydroxyacetone phosphate                                       10   U/ml    glycerol-3-phosphate dehydrogenase                                            (derived from rabbit muscle and                                               manufactured by Boehringer-Mannheim                                           GmbH, Germany)                                                   350  U/ml    D-3-hydroxybutyrate dehydrogenase                                             (derived from Pseudomonas sp.                                                 and manufactured by Toyobo Co., Ltd., Japan)                     ______________________________________                                    

Procedure

1 ml of the above reagent was placed in each of six cuvettes. To therespective cuvettes were individually added 50 μl each of six types ofaqueous acetoacetic acid solutions respectively having concentrations of0, 50, 100, 150, 200 and 250 μM. The resultant mixtures in therespective cuvettes were individually allowed to react at 37° C. Withrespect to each of respective samples taken from the reaction mixturesin the six cuvettes, absorbances at 340 nm were measured 3 minutes and 8minutes after the start of the reaction. Absorbance values obtained 3minutes and 8 minutes after the start of the reaction with respect tothe sample from the cuvette containing the 0 μM concentration solutionwere taken as reagent blanks, and subtracted from the respectiveabsorbance values obtained with respect to the samples from theremaining five cuvettes, which had different acetoacetic acidconcentrations of from 50 to 250 μM as mentioned above. Using theabsorption data of the samples from the remaining five cuvettes and thereagent blanks, a change in absorbance as between the above two timepoints was calculated with respect to each of the samples. Results areshown in FIG. 4, which demonstrates the presence of good linearity inthe relationship between the change (difference) in absorbance (withrespect to the reaction mixture, as between 3 and 8 minutes after thestart of the reaction) and the acetoacetic acid concentration.

Industrial Applicability

According to the determination method of the present invention, an errorin the quantitative determination of D-3-hydroxybutyric acid andacetoacetic acid can be minimized since two types of coenzymesexhibiting absorbances at different absorption wave-lengths are used.Further, the sensitivity of the determination method can be greatlyincreased due to the utilization of the enzymatic cycling reaction.Thus, the method of the present invention ensures rapidness and accuracyin the determination of D-3-hydroxybutyric acid and acetoacetic acid,even with the use of a small quantity of a biological sample.

We claim:
 1. A method for the quantitative determination of at least oneketone body selected from the group consisting of D-3-hydroxybutyricacid and acetoacetic acid, which consists essentially of:reacting abiological sample containing at least one ketone body selected from thegroup consisting of D-3-hydroxybutyric acid and acetoacetic acid with areagent comprising:(1) a D-3-hydroxybutyrate dehydrogenase whichutilizes the following coenzymes (i) and (ii):(i) a first coenzymeselected from the group consisting of thionicotinamide adeninedinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; and (3) B₁ ;said components (1), (2) and (3) participating in the following cyclingreaction: ##STR8## wherein A₁ is a thio-NADP compound, a thio-NADcompound, an NADP compound or an NAD compound; A₂ is a reduced form ofA₁ ; B₁ is a reduced NADP compound or a reduced NAD compound when A₁ isa thio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; and B₂ is an oxidized form of B₁, wherein the reactionof said biological sample with said reagent is conducted underconditions adapted for the enzymatically catalytic and cycling reaction,thereby effecting said cycling reaction; and measuring and correlating achange in the amount of A₂ or B₁ to the quantity of said at least oneketone body.
 2. The method according to claim 1, wherein said thio-NADPcompound is thionicotinamide adenine dinucleotide phosphate (thio-NADP)or thionicotinamide hypoxanthine dinucleotide phosphate.
 3. The methodaccording to claim 1, wherein said thio-NAD compound is thionicotinamideadenine dinucleotide (thio-NAD) or thionicotinamide hypoxanthinedinucleotide.
 4. The method according to claim 1, wherein said NADPcompound is selected from the group consisting of nicotinamide adeninedinucleotide phosphate (NADP), acetylpyridine adenine dinucleotidephosphate (acetyl-NADP), acetylpyridine adenine hypoxanthinedinucleotide phosphate and nicotinamide hypoxanthine dinucleotidephosphate (deamino-NADP).
 5. The method according to claim 1, whereinsaid NAD compound is selected from the group consisting of nicotinamideadenine dinucleotide (NAD), acetylpyridine adenine dinucleotide(acetyl-NAD), acetylpyridine adenine hypoxanthine dinucleotide andnicotinamide hypoxanthine dinucleotide (deamino-NAD).
 6. A method forthe quantitative determination of at least one ketone body selected fromthe group consisting of D-3-hydroxybutyric acid and acetoacetic acid,which consists essentially of:reacting a biological sample containing atleast one ketone body selected from the group consisting ofD-3-hydroxybutyric acid and acetoacetic acid with a reagentcomprising:(1) a D-3-hydroxybutyrate dehydrogenase as a firstdehydrogenase which utilizes the following coenzymes (i) and (ii):(i) afirst coenzyme selected from the group consisting of thionicotinamideadenine dinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; (3) at leastone coenzyme selected from B₁ and B₂ ; and (4) a second dehydrogenasewhich does not react with D-3-hydroxybutyric acid but acts to promotethe reaction for converting B₂ to B₁ in the following cycling reaction,in combination with a substrate for said second dehydrogenase: saidcomponents (1), (2), (3) and (4) participating in the following cyclingreaction: ##STR9## wherein A₁ is a thio-NADP compound, a thio-NADcompound, an NADP compound or an NAD compound; A₂ is a reduced form ofA₁ ; B₁ is a reduced NADP compound or a reduced NAD compound when A₁ isa thio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; B₂ is an oxidized form of B_(1;) and B₂ →B₁ representsan enzymatic reaction producing B₁ from coenzyme B₂ under the action ofsaid second dehydrogenase, wherein the reaction of said biologicalsample with said reagent is conducted under conditions adapted for theenzymatically catalytic and cycling reaction, thereby effecting thecycling reaction; and measuring and correlating a change in the amountof A₂ to the quantity of said at least one ketone body.
 7. The methodaccording to claim 6, wherein said thio-NADP compound isthionicotinamide adenine dinucleotide phosphate (thio-NADP) orthionicotinamide hypoxanthine dinucleotide phosphate.
 8. The methodaccording to claim 6, wherein said thio-NAD compound is thionicotinamideadenine dinucleotide (thio-NAD) or thionicotinamide hypoxanthinedinucleotide.
 9. The method according to claim 6, wherein said NADPcompound is selected from the group consisting of nicotinamide adeninedinucleotide phosphate (NADP), acetylpyridine adenine dinucleotidephosphate (acetyl-NADP), acetylpyridine adenine hypoxanthinedinucleotide phosphate and nicotinamide hypoxanthine dinucleotidephosphate (deamino-NADP).
 10. The method according to claim 6, whereinsaid NAD compound is selected from the group consisting of nicotinamideadenine dinucleotide (NAD), acetylpyridine adenine dinucleotide(acetyl-NAD), acetylpyridine adenine hypoxanthine dinucleotide andnicotinamide hypoxanthine dinucleotide (deamino-NAD).
 11. A method forthe quantitative determination of at least one ketone body selected fromthe group consisting of D-3-hydroxybutyric acid and acetoacetic acid,which consists essentially of:reacting a biological sample containing atleast one ketone body selected from the group consisting ofD-3-hydroxybutyric acid and acetoacetic acid with a reagentcomprising:(1) a D-3-hydroxybutyrate dehydrogenase as a firstdehydrogenase which utilizes the following coenzymes (i) and (ii):(i) afirst coenzyme selected from the group consisting of thionicotinamideadenine dinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and, nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) at least onecoenzyme selected from A₁ and A₂ ; (3) B₁ ; and (5) a thirddehydrogenase which does not react with D-3-hydroxybutyric acid but actsto promote the reaction for converting A₂ to A₁, in the followingcycling reaction, in combination with a substrate for said thirddehydrogenase: said components (1), (2), (3) and (5) participating inthe following cycling reaction: ##STR10## wherein A₁ is a thio-NADPcompound, a thio-NAD compound, an NADP compound or an NAD compound; A₂is a reduced form of A₁ ; B₁ is a reduced NADP compound or a reduced NADcompound when A₁ ; is a thio-NADP compound or a thio-NAD compound, or areduced thio-NADP compound or a reduced thio-NAD compound when A₁ is anNADP compound or an NAD compound; B₂ is an oxidized form of B₁ ; and A₂→A₁ represents an enzymatic reaction producing A₁ from coenzyme A₂ underthe action of said third dehydrogenase, wherein the reaction of saidbiological sample with said reagent is conducted under conditionsadapted for the enzymatically catalytic and cycling reaction, therebyeffecting the cycling reaction; and measuring and correlating a changein the amount of B₁ to the quantity of said at least one ketone body.12. The method according to claim 11, wherein said thio-NADP compound isthionicotinamide adenine dinucleotide phosphate (thio-NADP) orthionicotinamide hypoxanthine dinucleotide phosphate.
 13. The methodaccording to claim 11, wherein said thio-NAD compound isthionicotinamide adenine dinucleotide (thio-NAD) or thionicotinamidehypoxanthine dinucleotide.
 14. The method according to claim 11, whereinsaid NADP compound is selected from the group consisting of nicotinamideadenine dinucleotide phosphate (NADP), acetylpyridine adeninedinucleotide phosphate (acetyl-NADP), acetylpyridine adeninehypoxanthine dinucleotide phosphate and nicotinamide hypoxanthinedinucleotide phosphate (deamino-NADP).
 15. The method according to claim11, wherein said NAD compound is selected from the group consisting ofnicotinamide adenine dinucleotide (NAD), acetylpyridine adeninedinucleotide (acetyl-NAD), acetylpyridine adenine hypoxanthinedinucleotide and nicotinamide hypoxanthine dinucleotide (deamino-NAD).16. An analytical reagent for use in the quantitative determination ofat least one ketone body selected from the group consisting ofD-3-hydroxybutyric acid and acetoacetic acid, which comprises:1) aD-3-hydroxybutyrate dehydrogenase which utilizes the following coenzymes(i) and (ii):(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and (ii) a second coenzyme selected from the groupconsisting of nicotinamide adenine dinucleotide phosphate or its analog(NADP compound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; and (3) B₁,wherein A₁ is a thio-NADP compound, a thio-NAD compound, an NADPcompound or an NAD compound; B₁ is a reduced NADP compound or a reducedNAD compound when A₁ is a thio-NADP compound or a thio-NAD compound, ora reduced thio-NADP compound or a reduced thio-NAD compound when A₁ isan NADP compound or an NAD compound.
 17. An analytical reagent for usein the quantitative determination of at least one ketone body selectedfrom the group consisting of D-3-hydroxybutyric acid and acetoaceticacid, which comprises:(1) a D-3-hydroxybutyrate dehydrogenase as a firstdehydrogenase which utilizes the following coenzymes (i) and (ii):(i) afirst coenzyme selected from the group consisting of thionicotinamideadenine dinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; (3) at leastone coenzyme selected from B₁ (defined hereinbelow) and B₂ ; and (4) asecond dehydrogenase which does not react with D-3-hydroxybutyric acidbut acts to promote the reaction for converting B₂ to B₁, in combinationwith a substrate for said second dehydrogenase, wherein A₁ is athio-NADP compound, a thio-NAD compound, an NADP compound or an NADcompound; B₁ is a reduced NADP compound or a reduced NAD compound whenA₁ is a thio-NADP compound or a thio-NAD compound, or a reducedthio-NADP compound or a reduced thio-NAD compound when A₁ is an NADPcompound or an NAD compound; B₂ is an oxidized form of B₁.
 18. Ananalytical reagent for use in the quantitative determination of at leastone ketone body selected from the group consisting of D-3-hydroxybutyricacid and acetoacetic acid, which comprises:(1) a D-3-hydroxybutyratedehydrogenase as a first dehydrogenase which utilizes the followingcoenzymes (i) and (ii):(i) a first coenzyme selected from the groupconsisting of thionicotinamide adenine dinucleotide phosphate or itsanalog (thio-NADP compound) and thionicotinamide adenine dinucleotide orits analog (thio-NAD compound), and (ii) a second coenzyme selected fromthe group consisting of nicotinamide adenine dinucleotide phosphate orits analog (NADP compound) and nicotinamide adenine dinucleotide or itsanalog (NAD compound), and catalyzes the reversible reaction producingacetoacetic acid from D-3-hydroxybutyric acid as a substrate; (2) atleast one coenzyme selected from A₁ and A₂ ; and (3) B₁ ; (5) a thirddehydrogenase which does not react with D-3-hydroxybutyric acid but actsto promote the reaction for converting A₂ to A₁, in combination with asubstrate for said third dehydrogenase, wherein A₁ is a thio-NADPcompound, a thio-NAD compound, an NADP compound or an NAD compound; B₁is a reduced NADP compound or a reduced NAD compound when A₁ is athio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; A₂ is a reduced form of A₁.
 19. A method for thequantitative determination of D-3-hydroxybutyric acid, which consistsessentially of:treating a biological sample containingD-3-hydroxybutyric acid and acetoacetic acid with acetoacetatedecarboxylase to eliminate the acetoacetic acid; reacting the resultantbiological sample with a reagent comprising:(1) a D-3-hydroxybutyratedehydrogenase which utilizes the following coenzymes (i) and (ii):(i) afirst coenzyme selected from the group consisting of thionicotinamideadenine dinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; and (3) B₁ ;said components (1), (2) and (3) participating in the following cyclingreaction: ##STR11## wherein A₁ is a thio-NADP compound, a thio-NADcompound, an NADP compound or an NAD compound; A₂ is a reduced form ofA₁ ; B₁ is a reduced NADP compound or a reduced NAD compound when A₁ isa thio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; and B₂ is an oxidized form of B₁, wherein the reactionof said biological sample with said reagent is conducted underconditions adapted for the enzymatically catalytic and cycling reaction,thereby effecting the cycling reaction; and measuring and correlating achange in the amount of A₂ or B₁ to the quantity of said D-3hydroxybutyric acid.
 20. A method for the quantitative determination ofat least one ketone body selected from the group consisting ofD-3-hydroxybutyric acid and acetoacetic acid, which comprises:reacting abiological sample containing at least one ketone body selected from thegroup consisting of D-3-hydroxybutyric acid and acetoacetic acid with areagent comprising:(1) a D-3-hydroxybutyrate dehydrogenase whichutilizes the following coenzymes (i) and (ii):(i) a first coenzymeselected from the group consisting of thionicotinamide adeninedinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; and (3) B₁ ;said components (1), (2) and (3) participating in the following cyclingreaction: ##STR12## wherein A₁ is a thio-NADP compound, a thio-NADcompound, an NADP compound or an NAD compound; A₂ is a reduced form ofA₁ ; B₁ is a reduced NADP compound or a reduced NAD compound when A₁ isa thio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; and B₂ is an oxidized form of B₁, wherein the reactionof said biological sample with said reagent is conducted underconditions adapted for the enzymatically catalytic and cycling reaction,thereby effecting said cycling reaction; and measuring and correlating achange in the amount of A₂ or B₁ to the quantity of said at least oneketone body, wherein each of A₁ and B₁ is used in a concentration of0.02 to 100 mM or more, and the D-3-hydroxybutyrate dehydrogenase isused in a concentration of 5 to 1,000 U/ml or more.
 21. A method for thequantitative determination of at least one ketone body selected from thegroup consisting of D-3-hydroxybutyric acid and acetoacetic acid, whichcomprises:reacting a biological sample containing at least one ketonebody selected from the group consisting of D-3-hydroxybutyric acid andacetoacetic acid with a reagent comprising:(1) a D-3-hydroxybutyratedehydrogenase as a first dehydrogenase which utilizes the followingcoenzymes (i) and (ii):(i) a first coenzyme selected from the groupconsisting of thionicotinamide adenine dinucleotide phosphate or itsanalog (thio-NADP compound) and thionicotinamide adenine dinucleotide orits analog (thio-NAD compound), and (ii) a second coenzyme selected fromthe group consisting of nicotinamide adenine dinucleotide phosphate orits analog (NADP compound) and nicotinamide adenine dinucleotide or itsanalog (NAD compound), and catalyzes the reversible reaction producingacetoacetic acid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ;(3) at least one coenzyme selected from B₁ and B₂ ; and (4) a seconddehydrogenase which does not react with D-3-hydroxybutyric acid but actsto promote the reaction for converting B₂ to B₁ in the following cyclingreaction, in combination with a substrate for said seconddehydrogenase:said components (1), (2), (3) and (4) participating in thefollowing cycling reaction: ##STR13## wherein A₁ is a thio-NADPcompound, a thio-NAD compound, an NADP compound or an NAD compound; A₂is a reduced form of A₁ ; B₁ is a reduced NADP compound or a reduced NADcompound when A₁ is a thio-NADP compound or a thio-NAD compound, or areduced thio-NADP compound or a reduced thio-NAD compound when A₁ is anNADP compound or an NAD compound; B₂ is an oxidized form of B₁ ; and B₁→B₁ represents an enzymatic reaction producing B₁ from coenzyme B₂ underthe action of said second dehydrogenase, wherein the reaction of saidbiological sample with said reagent is conducted under conditionsadapted for the enzymatically catalytic and cycling reaction, therebyeffecting the cycling reaction; and measuring and correlating a changein the amount of A₂ to the quantity of said at least one ketone body,wherein A₁ is used in a concentration of 0.02 to 100 mM or more, atleast one coenzyme selected from B₁ and B₂ is used in a concentration of0.05 to 5000 μM or more, said D-3-hydroxybutyrate dehydrogenase as thefirst dehydrogenase is used in a concentration of 5 to 1000 U/ml ormore, the second dehydrogenase is used in a concentration which is 20times or more the Km value thereof for B₂, and the substrate for thesecond dehydrogenase is used in a stoichiometrically excess amount. 22.A method for the quantitative determination of at least one ketone bodyselected from the group consisting of D-3-hydroxybutyric acid andacetoacetic acid, which comprises:reacting a biological samplecontaining at least one ketone body selected from the group consistingof D-3-hydroxybutyric acid and acetoacetic acid with a reagentcomprising:(1) a D-3-hydroxybutyrate dehydrogenase as a firstdehydrogenase which utilizes the following coenzymes (i) and (ii):(i) afirst coenzyme selected from the group consisting of thionicotinamideadenine dinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and, nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) at least onecoenzyme selected from A₁ and A₂ ; (3) B₁ ; and (5) a thirddehydrogenase which does not react with D-3-hydroxybutyric acid but actsto promote the reaction for converting A₂ to A₁, in the followingcycling reaction, in combination with a substrate for said thirddehydrogenase:said components (1), (2), (3) and (5) participating in thefollowing cycling reaction: ##STR14## wherein A₁ is a thio-NADPcompound, a thio-NAD compound, an NADP compound or an NAD compound; A₂is a reduced form of A₁ ; B₁ is a reduced NADP compound or a reduced NADcompound when A₁ is a thio-NADP compound or a thio-NAD compound, or areduced thio-NADP compound or a reduced thio-NAD compound when A₁ is anNADP compound or an NAD compound; B₂ is an oxidized form of B₁ ; and A₂→A₁ represents an enzymatic reaction producing A₁ from coenzyme A₂ underthe action of said third dehydrogenase, wherein the reaction of saidbiological sample with said reagent is conducted under conditionsadapted for the enzymatically catalytic and cycling reaction, therebyeffecting the cycling reaction; and measuring and correlating a changein the amount of B₁ to the quantity of said at least one ketone body.wherein at least one coenzyme selected from A₁ and A₂ is used in aconcentration of 0.05 to 5000 μM or more, B₁ is used in a concentrationof 0.02 to 100 mM or more, said D-3-hydroxybutyrate dehydrogenase as thefirst dehydrogenase is used in a concentration of 5 to 1000 U/ml ormore, the third dehydrogenase is used in a concentration which is 20times or more the Km value thereof for A₂, and the substrate for thethird dehydrogenase is used in a stoichiometrically excess amount. 23.An analytical reagent for use in the quantitative determination of atleast one ketone body selected from the group consisting ofD-3-hydroxybutyric acid and acetoacetic acid, which comprises:1) aD-3-hydroxybutyrate dehydrogenase which utilizes the following coenzymes(i) and (ii):(i) a first coenzyme selected from the group consisting ofthionicotinamide adenine dinucleotide phosphate or its analog (thio-NADPcompound) and thionicotinamide adenine dinucleotide or its analog(thio-NAD compound), and (ii) a second coenzyme selected from the groupconsisting of nicotinamide adenine dinucleotide phosphate or its analog(NADP compound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; and (3) B₁ ;wherein A₁ is a thio-NADP compound, a thio-NAD compound, an NADPcompound or an NAD compound; B₁ is a reduced NADP compound or a reducedNAD compound when A₁ is a thio-NADP compound or a thio-NAD compound, ora reduced thio-NADP compound or a reduced thio-NAD compound when A₁ isan NADP compound or an NAD compound, and wherein each of A₁ and B₁ ispresent in a concentration of 0.02 to 100 mM or more, and theD-3-hydroxybutyrate dehydrogenase is present in a concentration of 5 to1,000 U/ml or more.
 24. An analytical reagent for use in thequantitative determination of at least one ketone body selected from thegroup consisting of D-3-hydroxybutyric acid and acetoacetic acid, whichcomprises:(1) a D-3-hydroxybutyrate dehydrogenase as a firstdehydrogenase which utilizes the following coenzymes (i) and (ii):(i) afirst coenzyme selected from the group consisting of thionicotinamideadenine dinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate; (2) A₁ ; (3) at leastone coenzyme selected from B₁ and B₂ ; and (4) a second dehydrogenasewhich does not react with D-3-hydroxybutyric acid but acts to promotethe reaction for converting B₂ to B₁, in combination with a substratefor said second dehydrogenase, wherein A₁ is a thio-NADP compound, athio-NAD compound, an NADP compound or an NAD compound; B₁ is a reducedNADP compound or a reduced NAD compound when A₁ is a thio-NADP compoundor a thio-NAD compound, or a reduced thio-NADP compound or a reducedthio-NAD compound when A₁ is an NADP compound or an NAD compound; B₂ isan oxidized form of B₁, and wherein A₁ is present in a concentration of0.02 to 100 mM or more, at least one coenzyme selected from B₁ and B₂ ispresent in a concentration of 0.05 to 5000 μM or more, saidD-3-hydroxybutyrate dehydrogenase as the first dehydrogenase is presentin a concentration of 5 to 1000 U/ml or more, the second dehydrogenaseis present in a concentration which is 20 times or more the Km valuethereof for B₂, and the substrate for the second dehydrogenase ispresent in a stoichiometrically excess amount.
 25. An analytical reagentfor use in the quantitative determination of at least one ketone bodyselected from the group consisting of D-3-hydroxybutyric acid andacetoacetic acid, which comprises:(1) a D-3-hydroxybutyratedehydrogenase as a first dehydrogenase which utilizes the followingcoenzymes (i) and (ii):(i) a first coenzyme selected from the groupconsisting of thionicotinamide adenine dinucleotide phosphate or itsanalog (thio-NADP compound) and thionicotinamide adenine dinucleotide orits analog (thio-NAD compound), and (ii) a second coenzyme selected fromthe group consisting of nicotinamide adenine dinucleotide phosphate orits analog (NADP compound) and nicotinamide adenine dinucleotide or itsanalog (NAD compound), and catalyzes the reversible reaction producingacetoacetic acid from D-3-hydroxybutyric acid as a substrate; (2) atleast one coenzyme selected from A₁ and A₂ ; and (3) B₁ ; (5) a thirddehydrogenase which does not react with D-3-hydroxybutyric acid but actsto promote the reaction for converting A₂ to A₁, in combination with asubstrate for said third dehydrogenase, wherein A₁ is a thio-NADPcompound, a thio-NAD compound, an NADP compound or an NAD compound; B₁is a reduced NADP compound or a reduced NAD compound when A₁ is athio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; A₂ is a reduced form of A₁, and wherein at least onecoenzyme₋₋ selected from A₁ and A₂ is present in a concentration of 0.05to 5000 μM or more, B₁ is present in a concentration of 0.02 to 100 mMor more, and said D-3-hydroxybutyrate dehydrogenase as the firstdehydrogenase is present in a concentration of 5 to 1000 U/ml or more,the third dehydrogenase is present in a concentration which is 20 timesor more the Km value thereof for A₂, and the substrate for the thirddehydrogenase is present in a stoichiometrically excess amount.
 26. Amethod for the quantitative determination of D-3-hydroxybutyric acid,which comprises:treating a biological sample containingD-3-hydroxybutyric acid and acetoacetic acid with acetoacetatedecarboxylase to eliminate the acetoacetic acid; reacting the resultantbiological sample with a reagent comprising:(1) a D-3-hydroxybutyratedehydrogenase which utilizes the following coenzymes (i) and (ii):(i) afirst coenzyme selected from the group consisting of thionicotinamideadenine dinucleotide phosphate or its analog (thio-NADP compound) andthionicotinamide adenine dinucleotide or its analog (thio-NAD compound),and (ii) a second coenzyme selected from the group consisting ofnicotinamide adenine dinucleotide phosphate or its analog (NADPcompound) and nicotinamide adenine dinucleotide or its analog (NADcompound), and catalyzes the reversible reaction producing acetoaceticacid from D-3-hydroxybutyric acid as a substrate;(2) A₁ ; and (3) B₁ ;said components (1), (2) and (3) participating in the following cyclingreaction: ##STR15## wherein A₁ is a thio-NADP compound, a thio-NADcompound, an NADP compound or an NAD compound; A₂ is a reduced form ofA₁ ; B₁ is a reduced NADP compound or a reduced NAD compound when A₁ isa thio-NADP compound or a thio-NAD compound, or a reduced thio-NADPcompound or a reduced thio-NAD compound when A₁ is an NADP compound oran NAD compound; and B₂ is an oxidized form of B₁, wherein the reactionof said biological sample with said reagent is conducted underconditions adapted for the enzymatically catalytic and cycling reaction,thereby effecting the cycling reaction; and measuring and correlating achange in the amount of A₂ or B₁ to the quantity of saidD-3-hydroxybutyric acid, wherein each of A₁ and B₁ is used in aconcentration of 0.02 to 100 mM or more, and the D-3-hydroxybutyratedehydrogenase is used in a concentration of 5 to 1,000 U/ml or more.